Iron triggers Tv PI4P5K proteostasis and Arf-mediated cell membrane trafficking to regulate PIP₂ signaling crucial for multiple pathogenic activities of the parasitic protozoan Trichomonas vaginalis

Trichomonas vaginalis is the etiologic agent of trichomoniasis, one of the most common non-viral sexually transmitted infections globally. Our previous work reported the role of phosphatidylinositol 4,5-bisphosphates (PIP₂) signaling in the actin-dependent pathogenicity of T. vaginalis. This study further demonstrated that iron transiently regulated T. vaginalis phosphatidylinositol-4-phosphate 5-kinase (TvPI4P5K) proteostasis and its complex formation with an active ADP ribosylation factor TvArf220, facilitating co-trafficking to the plasma membrane, crucial for PIP₂ production. In dominant-active HA-TvArf220 Q71L mutant, TvPI4P5K plasma membrane trafficking, PIP₂ production, and intracellular calcium levels were increased, while these processes were inhibited in dominant-negative T31N mutant or those by Brefeldin A (BFA) treatment. Additionally, PIP₂ replenishment reversed these inhibitions in the T31N mutant, suggesting the critical role of TvArf220 activation in PIP₂-calcium signaling. Also, T31N mutant and BFA treatment impaired actin dynamics and cytoskeleton-dependent processes in T. vaginalis, further linking the role of TvArf220 to PIP₂-calcium-dependent actin dynamics. Beyond cytoadherence, during host-parasite interactions, TvArf220 influenced both contact-dependent and -independent cytotoxicity, as well as phagocytotic capacity, contributing to the cytopathogenesis of human vaginal epithelial cells. Our findings underscore the key upstream regulation mechanisms of the PIP₂ signaling, orchestrating the interplay between TvArf220-PIP₂-calcium signaling and downstream actin cytoskeleton-driven pathogenicity in T. vaginalis.IMPORTANCETrichomonas vaginalis actin cytoskeleton-centric pathogenicity is regulated by the phosphatidylinositol 4,5-bisphosphates (PIP₂)-triggered calcium signaling cascade in response to environmental iron, though the detailed mechanism by which iron modulates PIP₂ signaling remains unclear. Our findings reveal that iron rapidly induces T. vaginalis phosphatidylinositol-4-phosphate 5-kinase (TvPI4P5K) translation followed by its degradation, while simultaneously activating TvArf220 binding, which facilitates TvPI4P5K localization to the plasma membrane for PIP₂ production. In contrast to the TvArf220 Q71L mutant, the reduced PIP₂ production, intracellular calcium, actin assembly, morphogenesis, and cytoadherence in the dominant-negative T31N mutant were recovered by PIP₂ supplementation, indicating the essential role of TvArf220 in PIP₂-dependent calcium signaling. Additionally, the contact-dependent or -independent cytotoxicity, along with the phagocytosis, was impaired in the TvPI4P5K- or TvArf220-deficient parasites, as well as in those treated with BAPTA or Latrunculin B. These findings highlight that TvArf220-mediated PIP₂-calcium signaling cascade regulates actin cytoskeleton and cytopathogenicity of T. vaginalis. This study uncovers a novel pathogenic mechanism and suggests potential therapeutic targets for parasite control.